Morphological observations and rates of protein synthesis in rat muscles incubated in vitro.

Isolated soleus and extensor digitorum longus muscles from small (40 or 70 g) rats developed a central and substantial (13-57%) loss of glycogen and alpha-glucan phosphorylase activity after incubation for up to 2 h in vitro. The central 'core' of the muscles showed a marked decrease in the rate of protein synthesis. It is suggested that during brief periods of incubation the central core of isolated rat muscles becomes hypoxic, and that consequently the viability of such muscles must be in question.     

Comparative rates of protein synthesis of rat kidney medulla and cortex in vitro

The $\text{in}\text{vitro}$ rate of ¹⁴C-leucine incorporation into protein has been examined in rat kidney tissue. The presence of a marked gradient was observed. Thus, the white medulla was the most active in this respect followed by, in descending order, red medulla and cortex. ¹⁴C-Leucine incorporation into protein was completely abolished in the presence of cycloheximide. The distribution of labeled protein between the medium and slice suggests a high degree of cellular integrity and little secretion of labeled protein from slice to medium. The pattern of ¹⁴C-leucine incorporation amongst the different zones of kidney of hypophysectomized rats was similar to that noted in normal rats.     

Differential synthesis by cultured atrial and ventricular rat cardiac myocytes of myosin light chain isoforms

Dissociated cells from neonatal rat atria and ventricles were cultured in monolayers for 3 days. Newly synthesized 35S-methionine labeled myosin light chain isoforms ALC-1, ALC-2 (atrial) and VLC-1, VLC-2 (ventricular) were identified on 2D gels, and their pattern of synthesis was compared to that of myocard fragments immediately after explanation. ALCs were synthesized in 5- to 10-fold excess over VLCs by atrial cultures, whereas the converse was true for ventricular cultures, with two exceptions: one third of the LC-1 synthesized by ventricular fragments was ALC-1, and dissociated atrial cells synthesized very little LC-2 of either isoform. The former finding corresponds to the relatively high proportion of ALC-1 in neonatal ventricular tissue. We conclude that the regional programme of LC isoform expression is basically retained after tissue explantation and even after dissociation and culturing of cardiac myocytes.     

Modeling atrial and ventricular contractility of the heart muscle. I. Modeling single contractions

A system of differential equations describing myocardium contractions in isometric and isotonic regimes has been obtained. On the basis of these equations functioning of myocardium was modelled on an electronic computer. An effect has been revealed of the coefficients of rheological equations and activation function on isometric and isotonic contraction of the force/rate ratio. A good agreement between the results of modelling and the experimental data has been observed.     

Ultrastructural localization of calsequestrin in adult rat atrial and ventricular muscle cells

The distribution of calsequestrin in rat atrial and ventricular myocardial cells was determined by indirect immunocolloidal gold labeling of ultrathin frozen sections. The results presented show that calsequestrin is confined to the sarcoplasmic reticulum where it is localized in the lumen of the peripheral and the interior junctional sarcoplasmic reticulum as well as in the lumen of the corbular sarcoplasmic reticulum, but absent from the lumen of the network sarcoplasmic reticulum. Comparison of these results with our previous studies on the distribution of the Ca2+ + Mg2+-dependent ATPase of the cardiac sarcoplasmic reticulum show directly that the Ca2+ + Mg2+-dependent ATPase and calsequestrin are confined to distinct regions within the continuous sarcoplasmic reticulum membrane. Assuming that calsequestrin provides the major site of Ca2+ sequestration in the lumen of the sarcoplasmic reticulum, the results presented support the idea that both junctional (interior and peripheral) and specialized nonjunctional (corbular) regions of the sarcoplasmic reticulum are involved in Ca2+ storage and possibly release. Furthermore, the structural differences between the junctional and the corbular sarcoplasmic reticulum support the possibility that Ca2+ storage and/or release from the lumen of the junctional and the corbular sarcoplasmic reticulum are regulated by different physiological signals.     

The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation.

Rates of protein synthesis were significantly lower in the cut soleus and extensor digitorum longus muscles than in their uncut counterparts. Rates of protein degradation were significantly higher in cut soleus muscles, but not in cut extensor digitorum longus muscles as compared with their uncut controls. Concentrations of ATP and phosphocreatine were significantly lower in cut soleus and extensor digitorum longus muscles after incubation in vitro in contrast with respective control uncut muscles. These data indicate that cutting of muscle fibres alters rates of protein synthesis and degradation, in addition to altering concentrations of high-energy phosphates. Since these findings stressed the importance of using intact muscles to study protein metabolism, additional studies were made on intact muscles in vitro. Stretched soleus muscles had higher concentrations of high-energy phosphates at the end of an incubation period than did unstretched muscles. However, the length of the soleus, extensor digitorum longus and diaphragm muscles during incubation did not affect rates of protein degradation.U     

Cytochrome c protein synthesis rate in rat skeletal muscle.

Endurance training is associated with increases in mitochondrial density, of which cytochrome c protein is an index. Increases in the synthesis rates of cytochrome c protein in skeletal muscle during endurance training have been inferred (Biochem. Biophys. Res. Commun. 66: 173, 1975; J. Biol. Chem. 252: 416, 1977). One purpose of the present study was to test these indirect approximations with direct measurements of the synthesis rates of cytochrome c protein in skeletal muscles postexercise. No change in the fractional synthesis rate of cytochrome c was detected in the red quadriceps muscle of rats either 2-7 h after a 104-min run on a motor-driven treadmill or 17-22 h after the final bout of 4 days of running 100 min/day. If the 16% increase in cytochrome c protein concentration in the red quadriceps muscle on the 5th day of training is used to calculate the nanomoles of cytochrome c synthesized per gram of wet muscle weight, the normalized rate of cytochrome c protein synthesis is increased 29% on the 5th day of training. The observation of no significant alteration in cytochrome c mRNA in the red quadriceps muscle of rats during the 1st wk of training implies that the initial increase in the synthesis rate of cytochrome c protein normalized per unit of muscle mass during treadmill training is likely to occur at a translational or posttranslational step. These results suggest that the control of increased cytochrome c expression in skeletal muscle during exercise training involves a complex mechanism.     

The metabolic state of muscle in the isolated perfused rat hemicorpus in relation to rates of protein synthesis.

Measures of perfusion adequacy in perfused rat hemicorpus preparations were investigated as potential indices of tissue function during studies of muscle protein metabolism. Perfusion under normal conditions for up to 80 min resulted in rates of protein synthesis and concentrations of ATP in muscle that were similar to those in vivo, but phosphocreatine in muscle gradually decreased and muscle lactate increased. Hypoxic conditions led to lower rates of protein synthesis, lower phospho-creatine and raised lactate contents in muscle compared with normal perfusions, and ATP was slightly decreased. Hypoxic preparations also released more lactate and K+ into the medium and had higher perfusion pressures, but glucose uptake and muscle water content were not altered. In totally ischaemic muscle, concentrations of ATP and phosphocreatine were even lower than in hypoxic muscle, and that of lactate was higher. From 11 preparations perfused for 60 min under normal conditions, three were selected on the basis of lower muscle ATP content than the others. Preparations with low ATP also showed lower muscle phosphocreatine concentrations, O2 uptake and CO2 output, as well as higher perfusion pressure and muscle lactate concentrations than in the remaining preparations, but muscle water, ADP and AMP concentrations and lactate and K+ flux were no different. In perfusions extended to 3 h, deterioration of function was more apparent. There were significant correlations between rates of protein synthesis and the concentrations of ATP, phosphocreatine and lactate in two different muscles (r = 0.756-0.929), but not with any of the other indices investigated. Taken overall, these experiments showed that concentrations of ADP, AMP and water in muscle, rates of lactate and glucose metabolism, K+ output, perfusion pressure and blood gas parameters were unsuitable for distinguishing unsound from sound preparations, because they did not consistently demonstrate differences, or could not be ascribed to only muscle metabolism. It was found that ATP, phosphocreatine and lactate concentrations in muscle were the best indicators of impaired metabolic state in studies of protein synthesis. Measurements of these could be used on a routine basis for rejecting unsatisfactory preparations.     

RNA bandages for photoregulating in vitro protein synthesis

'RNA bandages' are composed of two 6-12-mer 2'-OMe RNA strands complementary to a mRNA target that are joined by a photocleavable linker. These tandem oligonucleotides typically exhibit much higher affinity for the mRNA than the individual strands. An RNA bandage with binding arms of different lengths and a 4-base gap blocked translation in vitro of GFP mRNA; subsequent near-UV irradiation restored translation. This provides a general method of photomodulating hybridization for a variety of oligonucleotide-based technologies.     

DNA synthesis in cultures of atrial and ventricular cardiomyocytes in the rat

By means of 3H-thymidine autoradiography DNA replicative activity has been studied in cultured atrial and ventricular myocytes, and non-muscle cells from hearts of 2-week-old rats (age when cell proliferation in the myocardium is already significantly depressed). PAS-reaction was used as a cytochemical marker of cardiomyocytes: atrial myocytes are richer in glycogen than ventricular cells. Labeling indices of atrial myocytes after a 24 hour exposure to 3H-thymidine were higher than ventricular ones: on day 6 of culturing--47 and 5%, and on day 11-34 and 8%, respectively. After 10 days of culturing the number of binucleated atrial myocytes, non-typical for atrial myocardium in vivo, increased by 25-40% as compared with 8-13% on days 2-3 in culture. In 10-day cultures, 3- and 4-nucleated atrial myocytes were observed. Both mononucleated and binucleated atrial and ventricular myocytes incorporated 3H-thymidine. To find out whether the deeper inhibition of replicative activity in ventricular myocytes influences fibroblasts and endothelial cells from ventricles, the proliferative activity of non-muscle cells was studied. Non-muscle cells, both in atrial and ventricular cultures, behaved as a totally proliferating population (labeling indices on the 6th day are about 75-90%) and their growth rate decreased during the formation of the contact-inhibited monolayer. These cells, contrary to myocytes, are predominantly mononucleated in all the periods studied. The deeper depression of replication in ventricular myocytes appears to be related with their higher level of differentiation as compared to myocytes of the atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the synthesis rates of collagens and total protein in rabbit muscle.

1. Collagen- and total-protein-synthesis rates were determined in rabbit muscle by continuous infusion of radioactive proline. 2. The precursor pool of free proline used for collagen synthesis was defined by measuring the specific radioactivity of hydroxy-proline in isolated type I procollagen. The specific radioactivities of type I procollagen were about 40% of those for free proline in the homogenate. 3. The mean ratio (+/- S.E.M.) between the fractional synthesis rates of muscle collagen and total protein was 0.99 +/- 0.10, where the total protein values were based on specific radioactivities of the homogenate free proline pools. 4. Types I, III and V collagen were solubilized by pepsin and isolated by fractional precipitation with NaCl. The fractional synthesis rates of types I and III collagens were very similar. Type V collagen samples had higher specific radioactivities than the other collagens, but this was not necessarily indicative of a higher rate of synthesis because of uncertainty about the cellular origin of this collagen and, hence, the specific radioactivity of its precursor proline pool.     

Stimulation of protein synthesis in cultured heart muscle cells by glucose.

Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of alpha-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate.